The increasing prevalence of multiple drug resistance in pathogenic bacteria is a major threat to human health. Especially Gram-negative bacteria are often intrinsically resistant to a number of antimicrobial agents, and such intrinsic resistance may easily become increased by common mutations. Studies in our and other laboratories in recent years led to the conclusion that such resistance is frequently mediated by mechanisms preventing the access of agents to their targets inside bacterial cells. Two "barriers" work in a truly synergistic manner. First, the Gram-negative outer membrane greatly retards the entry of antimicrobial agents, both hydrophilic and lipophilic, because the narrowness or paucity of porin channels makes the entry of hydrophilic agents difficult, and because the lipopolysaccharide-phospholipid asymmetric bilayer of this membrane slows down drastically the entry of lipophilic agents. Second, a few molecules of antibiotics that succeed in crossing the outer membrane are actively pumped out by ubiquitous multidrug efflux pumps that often show an incredibly wide substrate specificity. The pumps are frequently overproduced in the multidrug- resistant strains from clinical sources; this mechanism is especially troublesome because the use of a single drug can lead to simultaneous resistance to most of the existing antibiotics. We will continue to study both phases of the access-prevention mechanism. In the area of outer membrane permeability, we will study the mechanism whereby some porins are folded to produce only a few open channels. We will also study the molecular mechanism which makes the lipopolysaccharide-phospholipid bilayer so exceptionally impermeable, and will measure, for the first time, the entry of aminoglycosides across the outer membrane. In the area of multidrug efflux pumps, we will continue our studies of the structure and function of the major multidrug efflux pump of Escherichia coli, AcrAB-TolC, to understand why this transporter can handle such a wide range of compounds. The results of these studies will be very useful for the development of synthetic and semisynthetic antimicrobial compounds in the future.